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Immune effector cell (IEC) therapies offer durable and sustained remissions in significant numbers of patients with hematological cancers. While these unique immunotherapies have improved outcomes for pediatric and adult patients in a number of disease states, as ‘living drugs,’ their toxicity profiles, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), differ markedly from conventional cancer therapeutics. At the time of article preparation, the US Food and Drug Administration (FDA) has approved tisagenlecleucel, axicabtagene ciloleucel, and brexucabtagene autoleucel, all of which are IEC therapies based on genetically modified T cells engineered to express chimeric antigen receptors (CARs), and additional products are expected to reach marketing authorization soon and to enter clinical development in due course. As IEC therapies, especially CAR T cell therapies, enter more widespread clinical use, there is a need for clear, cohesive recommendations on toxicity management, motivating the Society for Immunotherapy of Cancer (SITC) to convene an expert panel to develop a clinical practice guideline. The panel discussed the recognition and management of common toxicities in the context of IEC treatment, including baseline laboratory parameters for monitoring, timing to onset, and pharmacological interventions, ultimately forming evidence- and consensus-based recommendations to assist medical professionals in decision-making and to improve outcomes for patients.

Although many children and young adults with B-cell acute lymphoblastic leukemia (B-ALL) are cured with modern, risk-adapted chemotherapy regimens, 10% to 15% of patients will experience relapse or have refractory disease. Recent efforts to further intensify cytotoxic chemotherapy regimens in the frontline setting have failed as a result of excessive toxicity or lack of improvement in efficacy. As a result, novel approaches will be required to achieve cures in more newly diagnosed patients. Multiple immune-based therapies have demonstrated considerable efficacy in the setting of relapsed or refractory (R/R) disease, including CD19 targeting with blinatumomab and tisagenlecleucel and CD22 targeting with inotuzumab ozogamicin. These agents are now under investigation by the Children’s Oncology Group (COG) in clinical trials for newly diagnosed B-ALL, with integration into standard chemotherapy regimens based on clinically and biology-based risk stratification as well as disease response.

OBJECTIVE To characterize the incidence and clinical characteristics of neurotoxicity in the month following CTL019 infusion in children and young adults, to define the relationship between neurotoxicity and cytokine release syndrome (CRS), and to identify predictive biomarkers for development of neurotoxicity following CTL019 infusion. METHODS We analyzed data on 51 subjects, 4 to 22 years old, who received CTL019, a chimeric antigen receptor-modified T-cell therapy against CD19, between January 1, 2010 and December 1, 2015 through a safety/feasibility clinical trial (NCT01626495) at our institution. We recorded incidence of significant neurotoxicity (encephalopathy, seizures, and focal deficits) and CRS, and compared serum cytokine levels in the first month postinfusion between subjects who did and did not develop neurotoxicity. RESULTS Neurotoxicity occurred in 23 of 51 subjects (45%, 95% confidence interval = 31-60%) and was positively associated with higher CRS grade (p

INTRODUCTION: Cure rates for pediatric and young adult patients with refractory or recurrently relapsed acute lymphoblastic leukemia (ALL) are dismal. Survival from time of relapse is typically measured in weeks to months, and standard chemotherapy and currently approved targeted therapy achieve remission in less than a third of affected patients. To date, the only definitive curative therapy has been allogeneic hematopoietic stem cell transplant (HSCT). Advances in immunotherapy, with the introduction of chimeric antigen receptor T-cell therapies and the development of tisagenlecleucel, have changed the landscape. AREAS COVERED: This review will describe the pharmacology of tisagenlecleucel and summarize the clinical evidence for its use in the treatment of multiple-relapsed or refractory B-cell ALL (B-ALL). Also discussed are other immunotherapies for B-ALL as well as the most commonly-encountered toxicities and corresponding management strategies. EXPERT COMMENTARY: Early phase trials indicate that tisagenlecleucel significantly improves survival for patients with B-ALL that is refractory or in second or later relapse. In responding patients, remissions have been reported on the order of years, and thus, tisagenlecleucel may herald a dramatic shift in the treatment paradigm of this largely fatal disease.

Twenty-eight patients were maintained on subcutaneous immunoglobulin replacement for persistent B-cell aplasia and agammaglobulinemia following CD19-targeted chimeric antigen receptor T-cell therapy for B-cell lymphoblastic leukemia. Patients were transitioned from intravenous to subcutaneous immunoglobulin replacement at a median of 11.5 months (range, 4-20). Increasing serum IgG level was significantly associated with a lower rate of sinopulmonary infection (P = 0.0072). The median serum IgG level during infection-free periods was 1000 mg/dL (range, 720-1430), which was significantly higher than IgG levels in patients with sinopulmonary infections. As such, we recommend maintaining a goal IgG level > 1000 mg/dL to provide optimal protection.

BACKGROUND: In a single-center phase 1-2a study, the anti-CD19 chimeric antigen receptor (CAR) T-cell therapy tisagenlecleucel produced high rates of complete remission and was associated with serious but mainly reversible toxic effects in children and young adults with relapsed or refractory B-cell acute lymphoblastic leukemia (ALL). METHODS: We conducted a phase 2, single-cohort, 25-center, global study of tisagenlecleucel in pediatric and young adult patients with CD19+ relapsed or refractory B-cell ALL. The primary end point was the overall remission rate (the rate of complete remission or complete remission with incomplete hematologic recovery) within 3 months. RESULTS: For this planned analysis, 75 patients received an infusion of tisagenlecleucel and could be evaluated for efficacy. The overall remission rate within 3 months was 81%, with all patients who had a response to treatment found to be negative for minimal residual disease, as assessed by means of flow cytometry. The rates of event-free survival and overall survival were 73% (95% confidence interval [CI], 60 to 82) and 90% (95% CI, 81 to 95), respectively, at 6 months and 50% (95% CI, 35 to 64) and 76% (95% CI, 63 to 86) at 12 months. The median duration of remission was not reached. Persistence of tisagenlecleucel in the blood was observed for as long as 20 months. Grade 3 or 4 adverse events that were suspected to be related to tisagenlecleucel occurred in 73% of patients. The cytokine release syndrome occurred in 77% of patients, 48% of whom received tocilizumab. Neurologic events occurred in 40% of patients and were managed with supportive care, and no cerebral edema was reported. CONCLUSIONS: In this global study of CAR T-cell therapy, a single infusion of tisagenlecleucel provided durable remission with long-term persistence in pediatric and young adult patients with relapsed or refractory B-cell ALL, with transient high-grade toxic effects. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT02435849 .).

Background T-ALL/LBL represent a class of devastating hematologic cancers with high rates of relapse and mortality in both children and adults. Despite intensive multi-agent chemotherapy regimens, fewer than 50% of adults and 85% of children with T-ALL survive beyond five years. For those who relapse after initial therapy, salvage regimens induce remissions in only ~20-30% of cases, and survival is dismal. T-ALL/LBL is a genetically diverse group, but with universal overexpression of CD7, making this a suitable target for immunotherapy. Despite the success of CAR-T cells in B-cell malignancies, CAR-T cell development in T-cell malignancies has proven challenging due to fratricide and high risk of contamination of the genetically modified CAR-T product with the patient's malignant T cells. WU-CART-007 is a CD7-directed, genetically modified, allogeneic, 'off the shelf', fratricide-resistant chimeric antigen receptor (CAR) T-cell product for the treatment of CD7+ve hematologic malignancies. Methods This multicenter, open-label, dose-escalation, Phase 1/2 study (NCT#04984356) of WU-CART-007 in patients ≥ 12 years old, with relapsed or refractory T-ALL/LBL is designed to characterize the safety, tolerability, dose-limiting toxicities (DLTs), and maximum tolerated dose (MTD)/maximum administered dose (MAD; if no MTD defined) (Phase 1), and to investigate the preliminary anti-tumor activity, as measured by objective response rate (ORR) and duration of response (DOR) (Phase 2). Phase 1 is comprised of a dose escalation segment and will proceed according to a standard 3+3 design testing up to 4 dose levels from 1 to 9 x 10 8 cells. Adolescent patients, ages 12-17, will be eligible for enrollment in Phase 1 Dose Escalation beginning at Dose level 3 and 4, and during Phase 2 Cohort Expansion. Upon reaching the MTD and/or RP2D, the Phase 2 portion comprised of the cohort expansion segment will be launched. A Simon's optimal two-stage design will be implemented to enroll patients (an interim analysis for futility in the first stage and the final analysis in the second stage) for Phase 2 dose expansion cohort to confirm safety and explore preliminary efficacy. All patients will receive a single infusion of WU-CART-007 cells on day 1 following a lymphodepleting conditioning therapy consisting of fludarabine and cyclophosphamide on days -5 to -3. Patients will be hospitalized for a minimum of 7 days following WU-CART-007 administration. Response will be assessed on Cycle 1 Day 28 (± 1 days), and at Months 3, 6, 12, and 24, by bone marrow aspirate and biopsy and PET/CT if indicated. Response will be defined as per modified NCCN Guidelines Version 2.2020. Disclosures Ghobadi: Atara: Consultancy; Amgen: Consultancy, Research Funding; Wugen: Consultancy; Celgene: Consultancy; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding. Locke: Janssen: Consultancy, Other: Scientific Advisory Role; Kite, a Gilead Company: Consultancy, Other: Scientific Advisory Role, Research Funding; Iovance Biotherapeutics: Consultancy, Other: Scientific Advisory Role; Legend Biotech: Consultancy, Other; Novartis: Consultancy, Other, Research Funding; Takeda: Consultancy, Other; Wugen: Consultancy, Other; Cowen: Consultancy; Umoja: Consultancy, Other; Bluebird Bio: Consultancy, Other: Scientific Advisory Role; Calibr: Consultancy, Other: Scientific Advisory Role; BMS/Celgene: Consultancy, Other: Scientific Advisory Role; GammaDelta Therapeutics: Consultancy, Other: Scientific Advisory Role; Cellular Biomedicine Group: Consultancy, Other: Scientific Advisory Role; Amgen: Consultancy, Other: Scientific Advisory Role; Allogene Therapeutics: Consultancy, Other: Scientific Advisory Role, Research Funding; EcoR1: Consultancy; Emerging Therapy Solutions: Consultancy; Gerson Lehrman Group: Consultancy; Moffitt Cancer Center: Patents & Royalties: field of cellular immunotherapy. Maude: Wugen: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding. Davidson-Moncada: Wugen: Current Employment. Cooper: Wugen: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties; NeoImmune Tech: Patents & Royalties; RiverVest: Consultancy.

Introduction: The most common severe toxicity associated with chimeric antigen receptor T-cells targeting CD19 (CART19) is cytokine release syndrome (CRS; PMID: 29972754). Our group and others have published seminal observations on the biology of CRS through cytokine profiling, measuring a small number of analytes (PMID: 27076371, 33434058). Multiple biomarkers including interferon gamma (IFNG), IL-6, and IL-10 have been associated with the development of severe CRS in previous studies (PMID: 33434058). To date, the only biomarker predictive of the development of CRS prior to infusion has been disease burden. To obtain a more robust understanding of CRS biology, we performed comprehensive secretome profiling to measure more than 1400 serum analytes on serial serum samples collected from patients treated with the 41BB-containing CTL019 on two clinical trials. Methods: Serum from patients enrolled on two clinical trials of the CART19 product CTL019 (NCT01626495 & NCT02906371) were obtained serially from pre-infusion to one month post infusion. Patients were categorised as having "minimal" (no CRS, Grade 1, or Grade 2) or "severe" (Grade 3 or 4) CRS. The serum secretome was profiled using the Olink Explore 1536 Analysis platform (Olink, Upsala, Sweden). 1484 proteins were measured from serum via proximity extension assay (PEA) high-multiplex immunoassay. Differential expression analysis, correlation analyses and receiver operating characteristic (ROC) calculations were performed using R (version 4.0.4) in RStudio. Significance was based on a fold change of greater than 2 or less than -2 and a false discovery rate of less than 0.05 calculated using a Benjamini-Hochberg correction. Results: 26 patients (10 NCT01626495 & 16 NCT02906371) were included comprising 128 unique datapoints from baseline to 35 days post-infusion. Thirteen patients had minimal and 13 had severe CRS. Differentially expressed proteins between minimal and severe CRS at the peak timepoint are shown in (A; green represents IFNG responsive proteins). Not surprisingly, proteins involved in IL-6 and IFNG signalling were increased, including biomarkers of hemophagocytic lymphohistiocytosis (HLH) such as VSIG4, CXCL9, CXCL10, CD163. The IL-18 signalling axis was dysregulated at peak CRS in severe patients with markedly elevated IL18 and IL18BP, despite prior reports suggesting IL-18 up-regulation is unique to the late CRS seen with CART22 (PMID: 32925169). Soluble markers of checkpoint inhibition, including soluble PDL1 (CD274) and LAG3 were also highly elevated. Finally, biomarkers of endothelial damage, such as PLAT, TMSB10 and CALCA were significantly elevated in patients with severe CRS. Pathway analysis revealed significant dysregulation in targetable cytokine, chemokine, and signalling pathways (B). A volcano plot of differentially expressed proteins at pre-infusion (C) identified a single protein, MILR1, as a candidate biomarker that was highly differentially expressed in patients who would subsequently develop severe CRS. MILR1 expression decreased over time (D). An ROC of MILR1 as a predictor for development of severe CRS (E) demonstrated pre-infusion elevated MILR1 could accurately predict development of severe CRS (sensitivity 88%, specificity 97%, AUC=0.977). We identified correlates of MILR1 at pre-infusion and found that MILR1 correlated most highly with soluble FLT3 (R=0.86, p Conclusions: With comprehensive secretome profiling we made multiple novel insights into the biology of CRS after CART19 and identified several potentially targetable proteins and pathways that could mitigate severe CRS. Similar secretome profiling in patients who developed neurotoxicity will also be shown. We identified two novel pre-infusion biomarkers that demonstrate significant capacity to predict the development of severe CRS following CART19 infusion. The inverse relationship apparent between FLT3 and FLT3LG that persists over time is an important finding that implies a potential biological role for FLT3/FLT3 ligand in the development of severe CRS. Mechanistic studies exploring the role of MILR1 and FLT3 in the initiation of CRS are ongoing. Figure 1 Figure 1. Disclosures Lambert: Novartis, shionogi, argenx, Rigel, octapharma: Consultancy; Rigel, Novartis, Sysmex, octapharma: Research Funding. Bassiri: Kriya Therapeutics: Consultancy, Current holder of individual stocks in a privately-held company; Guidepoint Global: Consultancy. Levine: Vycellix: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Other: Co-Founder and equity holder; Ori Biotech: Membership on an entity's Board of Directors or advisory committees; Immusoft: Membership on an entity's Board of Directors or advisory committees; Immuneel: Membership on an entity's Board of Directors or advisory committees; Avectas: Membership on an entity's Board of Directors or advisory committees; Akron: Membership on an entity's Board of Directors or advisory committees; In8bio: Membership on an entity's Board of Directors or advisory committees. Maude: Wugen: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding. June: Tmunity, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Current equity holder in publicly-traded company; Novartis: Patents & Royalties; AC Immune, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Consultancy. Barrett: Tmunity Therapeutics: Current Employment. Grupp: Novartis, Kite, Vertex, and Servier: Research Funding; Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding. Teachey: Janssen: Consultancy; NeoImmune Tech: Research Funding; Sobi: Consultancy; BEAM Therapeutics: Consultancy, Research Funding.

Background: Anti-CD19 Chimeric Antigen Receptor (CAR) T cell therapy has emerged as a mainstay in the treatment of patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (ALL). While trials have shown encouraging survival rates, up to 50% of patients receiving CART eventually relapse. Identification of risk factors for subsequent relapse is crucial, as this could allow tailored surveillance and treatment approaches for high-risk patients, potentially leading to improved morbidity and mortality. Complete remission with incomplete hematologic recovery (CRi) has been associated with decreased remission duration and overall survival in acute leukemias. As count suppression following CAR T cell therapy is frequently seen, this study evaluated CRi as a prognostic marker for worse relapse-free (RFS) and overall survival (OS) after CAR T cell therapy in comparison to complete remission with hematologic recovery (CR). Methods: Patients with r/r ALL who achieved a complete morphologic remission after receiving anti-CD19 CAR T cell treatment with the 4-1BB-containing products CTL019 or humanized CART19 in the context of a clinical trial (NCT01626495, NCT02374333, NCT02228096, NCT02435849, NCT02906371) or commercial product (tisagenlecleucel) at Children's Hospital of Philadelphia from 4/2012-4/2019 were identified. Patients who received prior CAR therapy, and those with Trisomy 21 were excluded. Demographic, disease and treatment characteristics, and outcome data were abstracted from the medical record or clinical trial datasets. CR was defined as achieving a morphologic remission with both an absolute neutrophil count (ANC) ≥1,000/µL and a transfusion-independent platelet count ≥100,000/µL at any time between 27-33 days after CAR T cell infusion, whereas those achieving a morphologic remission without complete hematologic recovery were defined as CRi. RFS and OS were described for each cohort. Exposure-outcome association was assessed via the log-rank test and multivariable Cox proportional hazard regression. Results: Of the 206 patients included in the analysis, 104 (51%) achieved CR, 102 (49%) CRi. Forty patients (39%) met criteria for CRi with both ANC 25% bone marrow blasts (43% vs 19%) at infusion, whereas more CR patients were MRD-negative (50% vs 30%) at infusion (p=0.002). CRi patients were also more likely to have Grade 3/4 CRS (38% vs 6.7%). Median length of follow-up for patients with CR was 39 months (range 7-89), which was not statistically significantly different than for those patients with CRi (41 months, 11-98, p=0.875). There was no difference in RFS when stratified by hematologic recovery (Figure 1, p=0.2165), with RFS at 36 months for CR of 57% (47-69) and CRi of 46% (36-59). OS was significantly lower (Figure 2, p=0.0081) for those with CRi, with 36-month OS for CR of 81% (74-89), and for CRi of 63% (54-73). In multivariable analysis adjusting for sex, prior blinatumomab, relapse number, disease burden at infusion, and maximum CRS grade, CR was not associated with either RFS (HR 0.76 [95%CI 0.50-1.17] p=0.2182) or OS (HR 0.74 [95%CI 0.43-1.29] p=0.2908), in comparison to CRi, Table 2. Discussion: Complete remission with incomplete hematologic recovery, manifesting as neutropenia and/or thrombocytopenia, at the first disease assessment following CAR T cell infusion should not be regarded as a harbinger of relapse and demonstrates that patients with CRi have similar probability of durable remission without further therapy. Anticipatory guidance should be provided to patients, and their families, that CRi is more frequently seen in patients who experience high grade CRS, who have high disease burden at infusion, and who are treated in first relapse. Figure 1 Figure 1. Disclosures Callahan: Novartis: Speakers Bureau. Rheingold: Optinose: Other: Spouse's current employment; Pfizer: Research Funding. Grupp: Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Novartis, Kite, Vertex, and Servier: Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding. Maude: Wugen: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding.

Background: CAR-modified T cells targeting CD19 have produced remarkable responses in relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL); however, relapse continues to be a substantial challenge. CD19+ relapses, which account for 33-78% of relapses, are associated with loss of CAR T-cell surveillance due to short persistence. Thus, strategies to improve functional persistence to prevent and treat CD19+ relapsed disease are crucial. Here, we report our experience administering reinfusions of murine or humanized 4-1BB CD19 CAR T cells in an effort to prolong persistence in patients with demonstrated short persistence to mitigate relapse risk, treat CD19+ relapsed disease, and produce responses after nonresponse to initial CAR infusion. Methods: This analysis included patients aged Results: Among 229 CAR-naïve and 33 CAR-exposed patients treated with CD19 CAR between 2012-2020, 81 received ≥1 reinfusion (investigational CTL019, n=44; commercial tisagenlecleucel, n=11; huCART19, n=13 CAR-naïve and n=13 CAR-exposed). In addition, 18 patients received PD-1 blockade after their first (n=11) or subsequent (n=7) reinfusions. Indications for first reinfusion were peripheral BCR (CAR-naïve, n=32; CAR-exposed, n=6), hematogones (CAR-naïve, n=21; CAR-exposed, n=4), CD19+ MRD/relapse (CAR-naïve, n=10, CAR-exposed, n=0), and nonresponse to initial infusion (CAR-naïve, n=5, CAR-exposed, n=3). CRS grade ≥2 (Penn scale) occurred in 19 patients (grade 2, n=13; grade 3, n=4; grade 4, n=2). Grade 3-4 events only occurred in patients with active disease at time of reinfusion. Twenty-two patients had an inpatient admission within 30 days of first reinfusion, of which 7 required intensive care unit admission Among the 63 patients reinfused for relapse prevention, 33 (52%) had a CR at day 28. With a median duration of follow-up of 38 mos, 13 experienced a subsequent relapse (7 CD19+, 4 CD19-, 2 CD19-subset negative), 4 received alternative therapy or allogeneic hematopoietic stem cell transplantation (HSCT) in remission, and 16 remain in remission without further therapy at a median of 39 mos after first reinfusion. The median duration of B-cell aplasia was 8 mos (IQR 2-35) after reinfusion. Of the 30 with no response (NR), 10 had a subsequent CD19+ relapse, 15 received alternative therapy or HSCT, and 5 remain in remission without further therapy at a median of 43 mos after reinfusion. CIR and OS were not statistically significantly different between patients with CR or NR (CIR, p=0.26; OS, p=0.25) (Figure A-B). However, at 24 mos after reinfusion, CIR was 29% (95% CI, 11-44%) for CR compared to 61% (95% CI, 24-80%) for NR; OS was 90% (95% CI, 80-100%) for both groups. Of the 10 patients reinfused for relapse, 5 (50%) had a CR; 2 subsequently experienced a CD19+ relapse, 2 received an HSCT in remission, and 1 remains in remission without further therapy at 18 mos after reinfusion. Of the 8 patients reinfused for nonresponse to initial infusion, 7 were evaluable; none had a CR, and all died at a median of 2.5 mos after reinfusion. Conclusions: Reinfusion of CTL019/tisagenlecleucel or huCART19 is safe, may prolong B-cell aplasia in patients with short CAR persistence and reduce relapse risk, and can induce remissions in patients with CD19+ relapsed disease. Thus, reinfusion may provide an alternative to HSCT for short persistence. However, reinfusion is not effective for patients with nonresponse to initial CAR infusion. Figure 1 Figure 1. Disclosures Callahan: Novartis: Speakers Bureau. Rheingold: Optinose: Other: Spouse's current employment; Pfizer: Research Funding. June: Tmunity, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Current equity holder in publicly-traded company; Novartis: Patents & Royalties; AC Immune, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Consultancy. Grupp: Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Novartis, Kite, Vertex, and Servier: Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding. Maude: Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Wugen: Consultancy.

The impact of immunotherapy has grown exponentially in the past 5 years. Principle illustrations are encouraging results with engineered T cells expressing a chimeric antigen receptor (CAR). This experimental therapy is developing simultaneously in pediatric and adult clinical trials, making this field particularly relevant and exciting for pediatric oncologists.CAR-modified T cells targeting CD19 have produced dramatic antitumor responses in patients with relapsed/refractory B cell acute lymphoblastic leukemia. Clinical trials from several institutions, in both children and adults, using distinct CAR T cell products have demonstrated similar high complete remission rates of 61-93%, with durable remissions observed. Although the development of CARs for other malignancies has lagged behind, research into novel approaches to overcome inherent challenges is promising.Clinical trials of CAR-modified T cells have produced unprecedented results and are anticipated to have a broader impact as this approach expands into other indications, including other cancers and frontline therapy. The potential for long-term disease control, if fully realized, will have a transformative impact on the field.

Objective Initial success with chimeric antigen receptor-modified T cell therapy for relapsed/refractory acute lymphoblastic leukemia is leading to expanded use through multicenter trials. Cytokine release syndrome, the most severe toxicity, presents a novel critical illness syndrome with limited data regarding diagnosis, prognosis, and therapy. We sought to characterize the timing, severity, and intensive care management of cytokine release syndrome after chimeric antigen receptor-modified T cell therapy. Design Retrospective cohort study. Setting Academic children's hospital. Patients Thirty-nine subjects with relapsed/refractory acute lymphoblastic leukemia treated with chimeric antigen receptor-modified T cell therapy on a phase I/IIa clinical trial (ClinicalTrials.gov number NCT01626495). Interventions All subjects received chimeric antigen receptor-modified T cell therapy. Thirteen subjects with cardiovascular dysfunction were treated with the interleukin-6 receptor antibody tocilizumab. Measurements and main results Eighteen subjects (46%) developed grade 3-4 cytokine release syndrome, with prolonged fever (median, 6.5 d), hyperferritinemia (median peak ferritin, 60,214 ng/mL), and organ dysfunction. Fourteen (36%) developed cardiovascular dysfunction treated with vasoactive infusions a median of 5 days after T cell therapy. Six (15%) developed acute respiratory failure treated with invasive mechanical ventilation a median of 6 days after T cell therapy; five met criteria for acute respiratory distress syndrome. Encephalopathy, hepatic, and renal dysfunction manifested later than cardiovascular and respiratory dysfunction. Subjects had a median of 15 organ dysfunction days (interquartile range, 8-20). Treatment with tocilizumab in 13 subjects resulted in rapid defervescence (median, 4 hr) and clinical improvement. Conclusions Grade 3-4 cytokine release syndrome occurred in 46% of patients following T cell therapy for relapsed/refractory acute lymphoblastic leukemia. Clinicians should be aware of expanding use of this breakthrough therapy and implications for critical care units in cancer centers.

Background CART19-related toxicities may require treatment inpatient or in the ICU. We sought to describe inpatient/ICU resource utilization within 30 days of CART19 infusion and evaluate trends in resource utilization from 2012-2019. Methods We identified patients (pts) with ALL treated with CART19 on a clinical trial (NCT01626495, NCT02906371, and NCT02374333) or with the commercial product, tisagenlecleucel, at Children's Hospital of Philadelphia. Demographic, pharmacy, and inpatient data were extracted from the EHR from day of infusion (d0) to d+30 using a semi-automated EPIC data query tool. The Virtual Pediatric Systems database was queried for resource utilization data and PRISM 3 and PIM 2 severity of illness scores. Log-binomial and linear regression were used to estimate the association of patient characteristics with inpatient/ICU admission and inpatient/ICU length of stay (LOS). Similar models were used to estimate trends over time. Results The analyses included 213 pts. Median age was 12y (range 1-29y); 60% were male. Prior to CART19, 42% had an alloHCT. Pre-infusion, 19% had high tumor burden (HTB), defined as bone marrow blasts ≥40%. From 2012-2019, the proportion of pts with prior alloHCT or HTB decreased (Table 1). CART19 was infused in the outpatient setting in 198 (93%) pts. From d0 to d+30, 149 (70%) had ≥1 inpatient admission, starting at a median of d+2 (IQR +1 to +6). Among admitted pts, median cumulative inpatient LOS was 7d (IQR 4-13). From 2012-2019, there were linear trends toward decreases in proportion of pts admitted (p ICU admission was required for 49 (23%) pts, starting at a median of day +5 (IQR +4 to +7). ICU admission was more frequent for pts with HTB [HTB, 68% (95% CI, 52-81) vs. low burden, 11% (95% CI, 7-17), p Other than HTB, baseline characteristics were not associated with inpatient/ICU admission or LOS. Conclusion In a cohort of 213 pediatric pts, over 90% were safely infused with CART19 in the outpatient setting. Though 70% required at least one admission, the proportion of pts admitted to the hospital or ICU and cumulative inpatient LOS decreased over the past 7 years.

Tisagenlecleucel, a chimeric antigen receptor (CAR) T-cell product targeting CD19 is approved for relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, the impact of pretreatment variables, such as CD19 expression level, on leukemic blasts, the presence of CD19(–) subpopulations, and especially prior CD19-targeted therapy, on the response to CAR T-cell therapy has not been determined. We analyzed 166 patients treated with CAR T-cell therapy at our institution. Eleven patients did not achieve a minimal residual disease (MRD)(–) deep remission, whereas 67 patients had a recurrence after achieving a MRD(–) deep remission: 28 patients with CD19(+) leukemia and 39 patients with CD19(–) leukemia. Return of CD19(+) leukemia was associated with loss of CAR T-cell function, whereas CD19(–) leukemia was associated with continued CAR T-cell function. There were no significant differences in efficacy of CAR T cells in CD19-dim B-ALL, compared with CD19-normal or -bright B-ALL. Consistent with this, CAR T cells recognized and lysed cells with very low levels of CD19 expression in vitro. The presence of dim CD19 or rare CD19(–) events by flow cytometry did not predict nonresponse or recurrence after CAR T-cell therapy. However, prior therapy with the CD19-directed, bispecific T-cell engager blinatumomab was associated with a significantly higher rate of failure to achieve MRD(–) remission or subsequent loss of remission with antigen escape. Finally, immunophenotypic heterogeneity and lineage plasticity were independent of underlying clonotype and cytogenetic abnormalities.

Chimeric antigen receptor (CAR)-modified T-cell therapy has revolutionized the care of patients with relapsed and refractory B-cell acute lymphoblastic leukemia (B-ALL). Results from clinical trials across multiple institutions report remarkable remission rates with CD19-directed CAR-modified T-cell therapy. These remissions are also proving to be durable in many patients with a relapse-free survival (RFS) of approximately 50% to 60% at 1 year across several trials and institutions in this population that has been historically very difficult to treat. In addition, new products are being developed to enhance upon the original CAR T-cell products, which include a humanized CAR, allogeneic CARs, and both CD22 and biallelic CD19 and CD22 constructs. Toxicity after CAR-modified T-cell therapy is characterized by cytokine release syndrome (CRS) and neurotoxicity in the acute post-infusion period and B-cell aplasia as a long-term consequence of treatment. This review will summarize the published data thus far on the use of CAR-modified T-cell therapy in pediatric B-ALL and outline the various CAR products now being developed for this population. Delivery of this therapy and the decision to pursue hematopoietic stem cell transplant (HSCT) after treatment will be discussed.

While CD19-directed chimeric antigen receptor (CAR) T cells can induce remission in patients with B cell acute lymphoblastic leukemia (ALL), a large subset relapse with CD19- disease. Like CD19, CD22 is broadly expressed by B-lineage cells and thus serves as an alternative immunotherapy target in ALL. Here we present the composite outcomes of two pilot clinical trials ( NCT02588456 and NCT02650414 ) of T cells bearing a 4-1BB-based, CD22-targeting CAR in patients with relapsed or refractory ALL. The primary end point of these studies was to assess safety, and the secondary end point was antileukemic efficacy. We observed unexpectedly low response rates, prompting us to perform detailed interrogation of the responsible CAR biology. We found that shortening of the amino acid linker connecting the variable heavy and light chains of the CAR antigen-binding domain drove receptor homodimerization and antigen-independent signaling. In contrast to CD28-based CARs, autonomously signaling 4-1BB-based CARs demonstrated enhanced immune synapse formation, activation of pro-inflammatory genes and superior effector function. We validated this association between autonomous signaling and enhanced function in several CAR constructs and, on the basis of these observations, designed a new short-linker CD22 single-chain variable fragment for clinical evaluation. Our findings both suggest that tonic 4-1BB-based signaling is beneficial to CAR function and demonstrate the utility of bedside-to-bench-to-bedside translation in the design and implementation of CAR T cell therapies.

Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) is a genetically heterogeneous subtype of B-cell ALL characterized by chromosomal rearrangements and mutations that result in aberrant cytokine receptor and kinase signaling. In particular, chromosomal rearrangements resulting in the overexpression of cytokine receptor-like factor 2 (CRLF2) occur in 50% of Ph-like ALL cases. CRLF2 overexpression is associated with particularly poor clinical outcomes, though the molecular basis for this is currently unknown. Glucocorticoids (GCs) are integral to the treatment of ALL and GC resistance at diagnosis is an important negative prognostic factor. Given the importance of GCs in ALL therapy and the poor outcomes for patients with CRLF2 overexpression, we hypothesized that the aberrant signal transduction associated with CRLF2 overexpression might mediate intrinsic GC insensitivity. To test this hypothesis, we exposed Ph-like ALL cells from patient-derived xenografts to GCs and found that CRLF2 rearranged (CRLF2R) leukemias uniformly demonstrated reduced GC sensitivity in vitro. Furthermore, targeted inhibition of signal transduction with the MEK inhibitor trametinib and the Akt inhibitor MK2206, but not the JAK inhibitor ruxolitinib, was sufficient to augment GC sensitivity. These data suggest that suboptimal GC responses may in part underlie the poor clinical outcomes for patients with CRLF2 overexpression and provide rationale for combination therapy involving GCs and signal transduction inhibitors as a means of enhancing GC efficacy.

125 Background: Prostate specific membrane antigen (PSMA) is a highly expressed tumor-associated antigen potentially amenable to chimeric antigen receptor-modified T (CAR-T) cell therapy for castration-resistant prostate cancer (CRPC). However, a primary challenge to the success of CAR-T therapy in CRPC is the immunosuppressive microenvironment, characterized by high levels of TGFβ. The immunosuppressive functions of TGFβ can be inhibited in T cells using a dominant negative TGFβ receptor (TGFβRdn), thereby enhancing antitumor immunity. Methods: We conducted a first-in-human phase 1 clinical trial to evaluate the feasibility, safety and preliminary efficacy of PSMA-directed/TGFβ-insensitive CAR-T cells (CART-PSMA-TGFβRdn) in patients with metastatic CRPC (NCT03089203). In a 3+3 dose-escalation design, patients received a single dose of 1-3 x 107/m2 (Cohort 1) or 1-3 x 108/m2 (Cohort 2) CART-PSMA-TGFβRdn cells without lymphodepleting (LD) chemotherapy. In Cohort 3, one patient received 1-3 x 108/m2 CART-PSMA-TGFβRdn cells following a LD chemotherapy regimen of cyclophosphamide and fludarabine (Cy/Flu). In Cohort -3, three patients received 1-3 x 107/m2 CART-PSMA-TGFβRdn cells following Cy/Flu. Patients underwent metastatic tumor biopsies at baseline and on day 10 following treatment. Quantitative PCR of CART-PSMA-TGFβRdn DNA was performed at serial timepoints to evaluate for CAR-T expansion and persistence in peripheral blood and trafficking to target tissues. Multiplex cytokine analysis assessed CART-PSMA-TGFβRdn bioactivity. Results: Ten patients received CART-PSMA-TGFβRdn therapy across dose-level cohorts. All CART-PSMA-TGFβRdn infusion products met target transduction efficiency. Evaluation of CAR-T cellular kinetics demonstrated dose-dependent peripheral blood T cell expansion, as well as tumor tissue trafficking in post-treatment tumor biopsies. At Cohort 2 and above, 5 of 7 treated patients developed grade ≥2 cytokine release syndrome (CRS). Marked increases in inflammatory cytokines (IL-6, IL-15, IL-2, IFNγ) correlated with high-grade CRS events. One grade 5 adverse event (sepsis) occurred in Cohort 3. PSA decline was observed in 6 of 10 patients (median decline -33.2%, range -11.6% to -98.3%), and PSA30 response occurred in 4 of 10 patients (including one patient achieving PSA < 0.1 ng/mL). Conclusions: Adoptive cellular therapy with CART-PSMA-TGFβRdn is safe and feasible in patients with metastatic CRPC. A dose-dependent and lymphodepletion chemotherapy-dependent relationship was observed with CART-PSMA-TGFβRdn cell expansion, cytokine expression, CRS, and anti-tumor effect. Correlative cell trafficking and paired tumor Nanostring analyses will be presented. Future clinical investigations seek to enhance anti-tumor efficacy, while optimizing the therapeutic window. Clinical trial information: NCT03089203.

T cells expressing a CD19-specific chimeric antigen receptor (CAR19) are demonstrating remarkable efficacy in hematologic malignancies. Treatment is often associated with life-threatening cytokine release syndrome (CRS) which can be effectively treated with cytokine blockade using the antibodies, Siltuximab or Tocilizumab respectively targeting IL-6 or the IL-6 receptor. As IL-6 blockade is moving into the clinic for the treatment of CRS as well as IL-6-driven rheumatologic and malignant diseases, clinicians are utilizing serum cytokine panels more frequently to assess the effects of IL-6 inhibitors. It is paramount to ascertain whether levels obtained are accurate, especially as certain drugs may, in theory, affect quantification. We report the comparative quantification of IL-6 and sIL-6R using Luminex-based immunoassay kits from two vendors. Our results indicate good agreement of the commercial immunoassays in measurement of IL-6 but disagreement in quantitation of sIL-6R. We found that both Siltuximab and Tocilizumab can interfere with the measurement of their respective ligands using reagents from one vendor but not the second. This has significant implications for the analysis of IL-6 and sIL-6R pharmacokinetics analysis in Siltuximab or Tocilizumab-treated patients. We found that high levels of IL-6 can falsely reduce the measured levels of sIL-6R and high levels of sIL-6R can reduce levels of IL-6 when measured with some commercial assays. These data demonstrate the importance of assessing the impact of cytokine-blocking agents on accuracy of clinical biomarker assays in other diseases, as drugs targeting TNF-alpha, IL1B, and IL5 are being used more frequently in a large number of diseases.

Chimeric antigen receptor (CAR)–modified T cells with anti-CD19 specificity are a highly effective novel immune therapy for relapsed/refractory acute lymphoblastic leukemia. Cytokine release syndrome (CRS) is the most significant and life-threatening toxicity. To improve understanding of CRS, we measured cytokines and clinical biomarkers in 51 CTL019-treated patients. Peak levels of 24 cytokines, including IFNγ, IL6, sgp130, and sIL6R, in the first month after infusion were highly associated with severe CRS. Using regression modeling, we could accurately predict which patients would develop severe CRS with a signature composed of three cytokines. Results were validated in an independent cohort. Changes in serum biochemical markers, including C-reactive protein and ferritin, were associated with CRS but failed to predict development of severe CRS. These comprehensive profiling data provide novel insights into CRS biology and, importantly, represent the first data that can accurately predict which patients have a high probability of becoming critically ill. Significance: CRS is the most common severe toxicity seen after CAR T-cell treatment. We developed models that can accurately predict which patients are likely to develop severe CRS before they become critically ill, which improves understanding of CRS biology and may guide future cytokine-directed therapy. Cancer Discov; 6(6); 664–79. ©2016 AACR. See related commentary by Rouce and Heslop, p. 579. This article is highlighted in the In This Issue feature, p. 561

The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor–armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms. Significance: CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms. Cancer Discov; 5(12); 1282–95. ©2015 AACR. See related commentary by Jackson and Brentjens, p. 1238. This article is highlighted in the In This Issue feature, p. 1225

e22504 Background: Pediatric/YA pts with r/r B-NHL are rare and have heterogenous, aggressive histology and poor prognosis. We report early results for tisagenlecleucel (anti-CD19 CAR-T cell therapy) in pediatric/YA pts with r/r B-NHL. Methods: BIANCA (NCT03610724) is a phase 2, single-arm, global, open-label trial of tisagenlecleucel in pediatric/YA pts with CD19+ r/r B-NHL. Pts must have confirmed mature B-NHL r/r to ≥1 prior lines of therapy and no active CNS involvement. Primary endpoint is ORR. Secondary outcomes include DOR, EFS, safety and pharmacokinetics. Results: As of Nov 4, 2019, 8 pts were enrolled, of whom 4 had large B-cell lymphoma (LBCL), 3 Burkitt lymphoma (BL), and 1 gray zone lymphoma (GZL) (Table). Five pts had ≥2 lines of prior therapy. Suitable apheresis product was harvested in all 8 pts. Five pts were infused and 3 were pending infusion at data cut off. Product was successfully manufactured within specifications for all infused pts. Median time from enrollment to infusion was 33 days (range 30-67). All 5 pts have ≥28 days follow up; 2 pts have ≥3 months follow up (median [range] 85 days [69-97]). All 8 pts received bridging chemotherapy (including 1 pt who also had surgery and 1 who also had radiotherapy). Tisagenlecleucel dose range was 0.3-1.1 × 108 CAR+ viable T cells (weight-based: 0.9-1.7 × 106 CAR+ viable T cells/kg). Cmax (range: Cmax= 8520-14,200 copies/µg; time to Cmax= 2-21 days; n = 4) was within range of expansion observed in pediatric/YA acute lymphoblastic leukemia and adult diffuse LBCL. All 5 pts had CRS; no grade ≥3 CRS was recorded. Three pts had neurologic events, including 2 grade 3/4 events. One pt died due to disease progression. Conclusions: Pediatric/YA pts with r/r B-NHL (including BL) were successfully infused with tisagenlecleucel in the BIANCA trial with a manageable safety profile. Apheresis/manufacturing were feasible in this cohort of rapidly progressing disorders. Tisagenlecleucel was shown to expand in vivo. BIANCA provides the first systematic data on CAR-T cell therapy in highly aggressive, pediatric/YA B-NHL. Planned enrollment is 35 pts (26 infused and evaluable). Clinical trial information: NCT03610724. [Table: see text]

10511 Background: CNS relapse of B-ALL is difficult to treat after cranial radiation or multiple relapses. Durable remissions of relapsed/refractory (r/r) B-ALL have been seen with CD19 CAR T cells; however, most trials excluded patients with active CNS disease. As we observed CAR trafficking into the CSF, we hypothesized that CD19 CAR T cells could control CNS B-ALL. Methods: We identified children and young adults with r/r CNS B-ALL treated on 4 clinical trials of CD19 CAR T cells, CTL019 or CTL119. NCT01626495 and NCT02435849 excluded active CNS disease, while the former in an amendment as well as NCT02374333 and NCT02906371 permitted active CNS disease controlled on therapy. All trials permitted CNS disease that cleared and excluded bulky intracranial disease that did not improve. We analyzed outcomes (CR, RFS) and safety. Results: We identified 65 patients 1-29y (median 10y) with r/r CNS B-ALL (CNS+) of 182 treated with CTL019/CTL119. There were no differences in age, sex, history of SCT or neurologic comorbidities in the CNS+ and CNS- cohorts. CNS+ patients were more likely to be in ≥2nd relapse (74% vs 46%, p < 0.01), to have received cranial radiation (58% vs 11%, p < 0.01), to have detectable CNS disease (p = 0.02) and less bone marrow disease pre-infusion (p < 0.01). At 1 mo post infusion, 62 (95%) CNS+ and 110 (94%) CNS- patients were in CR; 1 in each cohort died of sequelae of CRS and was inevaluable for response. All patients with CNS disease detected pre-infusion cleared by mo 3, including 9 in the CNS+ cohort [5 CNS2 ( < 5 CSF WBC with blasts), 4 CNS3 ( > 5 CSF WBC with blasts or exam/imaging evidence)] and 8 in the CNS- cohort (isolated CNS2 status pre-infusion). There was no difference in RFS (p = 0.28) in the CNS+ and CNS- cohorts [24-mo RFS: 61% (95% CI 46-73%) and 60% (95% CI 48-70%)]. There were 4 CNS relapses in the CNS+ cohort, and 1 in the CNS- cohort. Encephalopathy rate and grade was similar in the CNS+ and CNS- cohorts (52% vs 40% any grade; 12% vs 11% grade 3/4; p = 0.41). There were no deaths due to neurotoxicity (NT) and no statistically significant differences in incidence or severity of any NT or CRS in the CNS+ and CNS- cohorts. Conclusions: The CD19 CAR T cell therapies CTL019/CTL119 are effective at clearing CNS disease and inducing durable remissions in children and young adults with r/r CNS B-ALL. CNS relapse rates are low ( < 3%). Most CD19 CAR T cell trials excluded patients with active CNS disease, primarily due to the risk of NT. We show that patients with r/r CNS B-ALL that is adequately controlled prior to infusion can be safely treated with CD19 CAR T cells, with no increased risk of NT. Clinical trial information: NCT01626495, NCT02435849, NCT02374333, NCT02906371.

Background Patients (pts) receiving CTL019 (tisagenlecleucel) for B-ALL with high tumor burden (HTB) are at high risk for developing severe CRS. Tocilizumab, an IL-6 receptor antibody, is a vital component of severe CRS management; however, its role in preventing severe CRS is not known. We sought to determine the effectiveness of preemptive tocilizumab (PT) administration in decreasing the rate of grade (gr) 4 CRS in HTB pts. Methods We conducted a pilot trial of risk-adapted PT after CTL019 (NCT02906371). HTB pts, defined as ≥40% bone marrow (BM) blasts immediately before infusion, received a single dose of PT for high persistent fever (2 temperatures ≥38.5C in 24hr). The primary endpoint was frequency of gr4 CRS (Penn scale), with an observed rate of ≤5/15 predefined as clinically meaningful. Secondary endpoints included complete remission (CR) rate and ICU length of stay (LOS). A comparator cohort with HTB who received standard CRS management (stdCRS) was identified from the initial CTL019 trial (NCT01626495). Results Characteristics of the PT (n=15) and stdCRS (n=26) HTB cohorts are shown in Table 1. All pts developed gr≥2 CRS; median time to fever was longer in the PT cohort [PT, 3d (IQR 2-9); stdCRS, 2d (IQR 1-7), p=0.03]. Gr4 CRS was observed in 4/15 (27%) pts in the PT cohort vs. 13/26 (50%) in the prior stdCRS cohort [RR 0.53 (95% CI, 0.21-1.34), p=0.18]. In the stdCRS cohort, gr4 CRS was associated with earlier onset of fever (p=0.04). In patients with earlier CRS onset (fever by day +4), gr4 CRS was observed in 4/9 (44%) vs. 13/21 (62%) in the PT and stdCRS cohorts [RR 0.72 (95% CI, 0.32-1.60), p=0.42]. Except for a trend toward fewer vasoactive days in the PT cohort, ICU LOS and resource utilization were not significantly different (Table 2). The CR rate at day 28 was similar in the PT and stdCRS cohorts (87% vs. 85%, p=1.00). Conclusion Risk-adapted PT administration reduced gr4 CRS, meeting the predefined study endpoint, without impacting the CR rate. A secondary comparison to a prior trial showed a clinically meaningful decrease in the rate of gr4 CRS from 50% to 27%; however, the analysis was not powered to detect a statistically significant difference. Ongoing analyses will evaluate CAR T cell expansion, duration of remission, and additional safety endpoints, including rates of neurotoxicity.

TO THE EDITOR: Genetically reprogrammed T-cell therapy is a novel treatment approach being investigated in various hematologic malignancies, including relapsed and refractory pediatric B-cell acute lymphoblastic leukemia (B-ALL), multiple myeloma, diffuse large B-cell lymphoma, and chronic

Immunotherapy with chimeric antigen receptor (CAR)-modified T cells targeting CD19 for pediatric acute lymphoblastic leukemia (ALL) has demonstrated significant efficacy. The principle toxicity is cytokine release syndrome with resultant hypotension. However, the spectrum of cardiovascular effects associated with CAR T cell therapy has not been systematically evaluated. We reviewed all patients who received CD19-directed CAR T cells at the Children's Hospital of Philadelphia between April 2012 and September 2016. The primary endpoint was hypotension-requiring inotropic support. Secondary endpoints included echocardiographic dysfunction at discharge and 6-month follow-up. Descriptive and univariate analyses were performed, and 98 encounters were included (55% male patients; mean age, 11.8 years [range, 1.7 to 27.1]); 98% had B-ALL. Before infusion 10 had cardiomyopathy and 1 had single-ventricle physiology. Primary endpoint occurred in 24 patients with mean onset 4.6 days (range, 1 to 9) after CAR T cell infusion, including 6 patients receiving milrinone. Worsened systolic function occurred in 10 patients; there were no cardiac-related deaths. Pretreatment factors associated with primary endpoint included higher pretreatment blast percentage on bone marrow biopsy (blast 25%: odds ratio, 15.5; 95% confidence interval, 5.1 to 47.1; P .001) and baseline lower ejection fraction (P = .019) or diastolic dysfunction (P = .021); neither pre-existing cardiomyopathy (P = .062), total body irradiation (P = .629), nor anthracycline dose (P = .444) were associated. At discharge, 7 patients had worsened echocardiographic function, but persistent dysfunction by the 6-month follow-up was rare. Pretreatment factors were not associated with persistent dysfunction at discharge. This is the first study to describe the cardiovascular effects of pediatric CAR T cell therapy. Although 10% had new systolic dysfunction after treatment, persistence was rare. Pretreatment blast count 25% or pre-existing cardiac dysfunction increased the risk for hypotension-requiring inotropic support; these patients may warrant close observation.

We report a patient relapsing 9 months after CD19-targeted CAR T cell (CTL019) infusion with CD19(-) leukemia that aberrantly expressed the anti-CD19 CAR. The CAR gene was unintentionally introduced into a single leukemic B cell during T cell manufacturing, and its product bound in cis to the CD19 epitope on the surface of leukemic cells, masking it from recognition by and conferring resistance to CTL019.

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, affecting approximately 2900 children and adolescents in the United States each year (Ries et al., SEER Program, 1999). Although survival has improved from less than 10% a half century ago to almost 90% today, ALL remains the leading cause of pediatric cancer deaths (Hunger et al., J Clin Oncol 30(14):1663–9, 2012; Smith et al., J Clin Oncol 28(15):2625–34, 2010). From the first introduction of single-agent chemotherapy in the 1940s, combination chemotherapy and intensification of post-induction therapy have largely been responsible for advances in the treatment of ALL, as has therapy directed at the central nervous system (CNS). Over time, the stratification of patients by their risk of relapse, such that therapy intensity is tailored to this risk, has been a major contributor to both improvements in survival rates and reductions in toxicity from therapy. Clinical features, leukemia biology, and early response to therapy have emerged as important components of risk stratification. The biology of ALL, key prognostic factors, and treatment approaches will be discussed, with an emphasis on new developments in the treatment of pediatric ALL.

Background & Aim The most common side effect of CD19-directed CAR-T cell therapy is CRS. IL-6 blockade by toci typically results in rapid resolution of severe CRS in CTL019 treated pts; however, the ideal timing of toci remains unclear. Pts with high tumor burden (TB; defined as ≥40% marrow blasts) are at high risk for severe CRS. We hypothesized that preemptive toci administration early in the course of CRS in pts with high TB might decrease acute CRS severity without compromising efficacy. Methods, Results & Conclusion MTD We conducted a two-cohort, open-label study of the timing of toci on CTL019-associated CRS. Pts were 1-24 yo with CD19-expressing relapsed or refractory B-cell acute lymphoblastic leukemia (r/r B-ALL). CTL019 was manufactured at the UPenn Cell and Vaccine Production Facility. Enrolled pts were assigned to high (≥ 40%) or low ( 38.5 C measured twice in 24 hours (“early toci”) and subsequently treated per standard CRS algorithm. Primary endpoint: frequency of gr4 CRS (Penn scale) in ≤5/15 pts. Secondary endpoints: D28 tumor response, remission duration, additional toxicities. RES 78 pts were screened, all were enrolled. 65 pts were evaluable for analysis at data cutoff. 15 pts were assigned to HTBC, 50 pts to LTBC and all were infused with CTL019 (median dose 5.0 × 106 [range 5.6 × 105-3.9 × 107] CAR+ T cells/kg). The overall rate of gr4 CRS was 9% (27% in the HTBC, 4% in the LTBC, Table 1). The overall rate of gr1-4 CRS was 72% (100% in the HTBC, 64% in the LTBC). One pt in the HTBC died from CNS hemorrhage after gr4 CRS. All patients in the HTBC received the early toci intervention. The overall rate of gr3 and 4 CRS was lower than in two prior studies of CTL019 therapy in pediatric r/r B-ALL (18% vs 42% in CHP959 and 48% in ELIANA). The ORR at day 28 follow-up was 97% (87% HTBC, 100% LTBC, with 71% CR and 26% CRi, Table 2). CONC We defined clincially meaningful reduction in severe CRS if ≤5/15 subjects had gr4 CRS in the HTBC; this a priori defined primary end-point was met. Early administration of toci targeted to pts with high TB may reduce the incidence of severe CRS while maintaining excellent efficacy of CTL019 therapy. TB remains a major predictor of toxicity. Longer follow-up is ongoing to assess impact on duration of remission.

Background CD19-targeted chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable clinical efficacy in treating relapsed/refractory B-cell acute lymphoblastic leukemia (ALL). However, there are potential treatment-related toxicities, which may be severe. Acute kidney injury (AKI) has been reported after CD19 CAR T cells, but not systematically evaluated. We sought to describe AKI incidence, severity, outcome, and risk factors in the first 30 days after CTL019, a CD19 CAR T cell therapy, for ALL in pediatric patients. Methods We studied patients treated with CTL019 through two clinical trials (NCT01626495 and NCT02906371) at Children's Hospital of Philadelphia between April 2012 and April 2018. Demographic, laboratory and pharmacy data were automatically extracted from the electronic medical record using an EPIC data query tool. The primary outcome was AKI within 30 days after CTL019 infusion. AKI was defined using the Kidney Disease: Improving Global Outcomes criteria. Stage 1 (serum creatinine (SCr) >= 1.5 times the baseline) was classified as mild AKI. Stage 2 or 3 (SCr >=2 times the baseline) were classified as severe AKI. Renal recovery was defined as improvement in SCr to within 1.5 times the baseline by day +30. Log-binomial regression was used to estimate risk ratios for the association of cytokine release syndrome (CRS) and other patient characteristics with the development of AKI. Results A total of 125 patients and 3231 creatinine values were analyzed. Median patient age was 11.2 (range: 1.4-29.1) years at CTL019 infusion; 57.6% were male, 72% were Caucasian, and 82.4% were non-Hispanic. AKI developed in 26 patients (21.0%; 95% CI, 14.5 to 28.9), severe AKI developed in 15 patients (12%; 95% CI 7.3 to 19.1), and 3 patients (2.4%; 95% CI 0.7 to 7.3) required renal-replacement therapy. Among patients who developed AKI, 22 (88%: 95% CI 66.7 to 96.4) recovered renal function by day +30. Patients with Grade 3/4 CRS had a 4.9 times greater risk of developing AKI (95% CI, 2.4 to 9.9; p Conclusion In the first 30 days after CTL019 infusion, 21% of patients developed AKI, but most recovered renal function by day +30. AKI was strongly associated with Grades 3 and 4 CRS and developed at a median of 5 days after the start of CRS. Additional analyses will compare the trajectories of CRS biomarkers and tumor lysis labs with the trajectory of AKI in order to better elucidate mechanisms of renal injury and identify opportunities for intervention.

Adoptive transfer of T cells genetically modified to express chimeric antigen receptors (CARs) targeting the CD19 B cell-associated protein have demonstrated potent activity against relapsed/refractory B-lineage acute lymphoblastic leukemia (B-ALL). Not all patients respond, and CD19-negative relapses have been observed. Overexpression of the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) occurs in a subset of adults and children with B-ALL and confers a high risk of relapse. Recent data suggest the TSLPR signaling axis is functionally important, suggesting that TSLPR would be an ideal immunotherapeutic target. We constructed short and long CARs targeting TSLPR and tested efficacy against CRLF2-overexpressing B-ALL. Both CARs demonstrated activity in vitro, but only short TSLPR CAR T cells mediated leukemia regression. In vivo activity of the short CAR was also associated with long-term persistence of CAR-expressing T cells. Short TSLPR CAR treatment of mice engrafted with a TSLPR-expressing ALL cell line induced leukemia cytotoxicity with efficacy comparable with that of CD19 CAR T cells. Short TSLPR CAR T cells also eradicated leukemia in 4 xenograft models of human CRLF2-overexpressing ALL. Finally, TSLPR has limited surface expression on normal tissues. TSLPR-targeted CAR T cells thus represent a potent oncoprotein-targeted immunotherapy for high-risk ALL.

Early T-cell precursor (ETP) acute lymphoblastic leukemia (ALL) is a recently described subtype of T-ALL characterized by a unique immunophenotype and genomic profile, as well as a high rate of induction failure. Frequent mutations in cytokine receptor and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways led us to hypothesize that ETP-ALL is dependent on JAK/STAT signaling. Here we demonstrate aberrant activation of the JAK/STAT pathway in ETP-ALL blasts relative to non-ETP T-ALL. Moreover, ETP-ALL showed hyperactivation of STAT5 in response to interleukin-7, an effect that was abrogated by the JAK1/2 inhibitor ruxolitinib. In vivo, ruxolitinib displayed activity in 6 of 6 patient-derived murine xenograft models of ETP-ALL, with profound single-agent efficacy in 5 models. Ruxolitinib treatment decreased peripheral blast counts relative to pretreatment levels and compared with control (P < .01) in 5 of 6 ETP-ALL xenografts, with marked reduction in mean splenic blast counts (P < .01) in 6 of 6 samples. Surprisingly, both JAK/STAT pathway activation and ruxolitinib efficacy were independent of the presence of JAK/STAT pathway mutations, raising the possibility that the therapeutic potential of ruxolitinib in ETP-ALL extends beyond those cases with JAK mutations. These findings establish the preclinical in vivo efficacy of ruxolitinib in ETP-ALL, a biologically distinct subtype for which novel therapies are needed.

Relapsed and refractory leukemias pose substantial challenges in both children and adults, with very little progress being made in more than a decade. Targeted immunotherapy using chimeric antigen receptor (CAR)-modified T cells has emerged as a potent therapy with an innovative mechanism. Dramatic clinical responses with complete remission rates as high as 90% have been reported using CAR-modified T cells directed against the B-cell-specific antigen CD19 in patients with relapsed/refractory acute lymphoblastic leukemia. Supraphysiologic T-cell proliferation, a hallmark of this therapy, contributes to both efficacy and the most notable toxicity, cytokine release syndrome, posing a unique challenge for toxicity management. Further studies are necessary to identify additional targets, standardize approaches to cytokine release syndrome management, and determine the durability of remissions.

Immunotherapy for the treatment of cancer has advanced at a tremendous pace over the last decade. In this review, we provide an overview of recent progress in immunotherapy for the treatment of leukemia, focusing on antibody-drug conjugates (ADC), bi-specific T-cell engagers (BiTE), and chimeric antigen receptor (CAR) T cells.Ongoing clinical trials of CAR T cells directed against CD19 have produced complete remission rates as high as 93%, prompting global multicenter phase 2 trials and the first FDA approval of a CAR T-cell therapy. Insights into cytokine release syndrome, a toxicity of CAR T-cell therapy, and the cause for relapse after CAR T-cell therapy are evolving. The bispecific antibody blinatumomab and the ADCs inotuzumab and gemtuzumab have also recently received FDA approval for ALL and AML, respectively, moving these agents into a more prominent role in the relapse setting.The use of immunotherapy for leukemia has been successful in creating durable remissions for multiply relapsed and refractory patients who previously had little chance of cure. The ongoing clinical and preclinical work continues to advance our understanding of these immune-based therapies, and will shape the next generation of clinical trials.

As a consequence of acquired or intrinsic disease resistance, the prognosis for patients with relapsed or refractory T-cell acute lymphoblastic leukemia (T-ALL) is dismal. Novel, less toxic drugs are clearly needed. One of the most promising emerging therapeutic strategies for cancer treatment is targeted immunotherapy. Immune therapies have improved outcomes for patients with other hematologic malignancies including B-cell ALL; however no immune therapy has been successfully developed for T-ALL. We hypothesize targeting CD38 will be effective against T-ALL. We demonstrate that blasts from patients with T-ALL have robust surface CD38 surface expression and that this expression remains stable after exposure to multiagent chemotherapy. CD38 is expressed at very low levels on normal lymphoid and myeloid cells and on a few tissues of nonhematopoietic origin, suggesting that CD38 may be an ideal target. Daratumumab is a human immunoglobulin G1κ monoclonal antibody that binds CD38, and has been demonstrated to be safe and effective in patients with refractory multiple myeloma. We tested daratumumab in a large panel of T-ALL patient-derived xenografts (PDX) and found striking efficacy in 14 of 15 different PDX. These data suggest that daratumumab is a promising novel therapy for pediatric T-ALL patients.

Tisagenlecleucel (CTL019) is an investigational immunotherapy that involves reprogramming a patient's own T cells with a transgene encoding a chimeric antigen receptor to identify and eliminate CD19-expressing cells. We previously reported that CTL019 achieved impressive clinical efficacy in patients with relapsed/refractory B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), including the expansion and persistence of CTL019 cells, which correlates with response to therapy. Here, we performed formal cellular kinetic analyses of CTL019 in a larger cohort of 103 patients treated with CTL019 in 2 different diseases (ALL and CLL). CTL019 was measured in peripheral blood and bone marrow, using quantitative polymerase chain reaction and flow cytometry. CTL019 levels in peripheral blood typically peaked at 10 to 14 days postinfusion and then declined slowly over time. Patients with complete response (CR)/CR with incomplete count recovery had higher levels of CTL019 in peripheral blood, with greater maximal concentration and area under the curve values compared with nonresponding patients (P < .0001 for each). CTL019 transgene levels were measurable up to 780 days in peripheral blood. CTL019 trafficking and persistence were observed in bone marrow and cerebrospinal fluid. CTL019 expansion correlated with severity of cytokine release syndrome (CRS) and preinfusion tumor burden in pediatric ALL. The results described here are the first detailed formal presentation of cellular kinetics across 2 diseases and highlight the importance of the application of in vivo cellular kinetic analyses to characterize clinical efficacy and CRS severity associated with CTL019 therapy.

Relapsed acute lymphoblastic leukemia (ALL) is difficult to treat despite the availability of aggressive therapies. Chimeric antigen receptor-modified T cells targeting CD19 may overcome many limitations of conventional therapies and induce remission in patients with refractory disease.We infused autologous T cells transduced with a CD19-directed chimeric antigen receptor (CTL019) lentiviral vector in patients with relapsed or refractory ALL at doses of 0.76×10(6) to 20.6×10(6) CTL019 cells per kilogram of body weight. Patients were monitored for a response, toxic effects, and the expansion and persistence of circulating CTL019 T cells.A total of 30 children and adults received CTL019. Complete remission was achieved in 27 patients (90%), including 2 patients with blinatumomab-refractory disease and 15 who had undergone stem-cell transplantation. CTL019 cells proliferated in vivo and were detectable in the blood, bone marrow, and cerebrospinal fluid of patients who had a response. Sustained remission was achieved with a 6-month event-free survival rate of 67% (95% confidence interval [CI], 51 to 88) and an overall survival rate of 78% (95% CI, 65 to 95). At 6 months, the probability that a patient would have persistence of CTL019 was 68% (95% CI, 50 to 92) and the probability that a patient would have relapse-free B-cell aplasia was 73% (95% CI, 57 to 94). All the patients had the cytokine-release syndrome. Severe cytokine-release syndrome, which developed in 27% of the patients, was associated with a higher disease burden before infusion and was effectively treated with the anti-interleukin-6 receptor antibody tocilizumab.Chimeric antigen receptor-modified T-cell therapy against CD19 was effective in treating relapsed and refractory ALL. CTL019 was associated with a high remission rate, even among patients for whom stem-cell transplantation had failed, and durable remissions up to 24 months were observed. (Funded by Novartis and others; CART19 ClinicalTrials.gov numbers, NCT01626495 and NCT01029366.).

Chimeric antigen receptor (CAR)-modified T cells and bispecific T cell-engaging antibodies have demonstrated dramatic clinical responses in recent clinical trials. The hallmark of these novel highly active immunotherapies is nonphysiologic T cell activation, which has correlated not only with greatly increased efficacy but also with notable toxicity in some cases. We and others have observed a cytokine release syndrome (CRS), which correlates with both toxicity and efficacy in patients receiving T cell-engaging therapies. In addition to elevations in effector cytokines, such as interferon-γ, cytokines associated with hemophagocytic lymphohistiocytosis or macrophage activation syndrome, such as interleukin (IL)-10 and IL-6, may also be markedly elevated. Whereas corticosteroids may control some of these toxicities, their potential to block T cell activation and abrogate clinical benefit is a concern. Detailed studies of T cell proliferation and the resultant immune activation produced by these novel therapies have led to more targeted approaches that have the potential to provide superior toxicity control without compromising efficacy. One approach we have developed targets IL-6, a prominent cytokine in CRS, using the IL-6R antagonist tocilizumab. We will review the pathophysiology and management options for CRS associated with T cell-engaging therapies.

Advances in treatment regimens have improved outcomes for children with acute myeloid leukemia (AML) over the past several decades with 5-year mortality rates decreasing from over 80% in the mid-1970s to approximately 40–60% currently (1). These improvements are largely due to intensification of chemotherapy regimens, which carries the risk of increased treatment-related morbidity and mortality (2–5). Data suggest that children treated for AML are at high risk for sepsis, respiratory failure requiring mechanical ventilation, and circulatory shock requiring inotropic support (2, 6, 7). Initial reports of children with cancer receiving mechanical ventilation showed ICU mortality rates as high as 70–74% (8, 9). These outcomes led to wide variations in practice, presumably due to concerns regarding the appropriateness of offering intensive care to children with cancer (10–12). Although more recent studies have shown lower mortality rates in children with cancer receiving mechanical ventilation (25–65%) (13–22), these rates are substantially higher than the reported 7.2% mortality rate for a general population of mechanically ventilated children (23). Notably, among children with cancer who receive both mechanical ventilation and inotropic support, reported mortality rates have ranged from 54% to 69% (20, 24, 25). Despite the intensity of modern AML treatment regimens, there are few contemporary data documenting the frequency of ICU care, risk of specific organ failures, and mortality in admissions requiring ICU care within this patient population. A single-center study of hospital resource utilization in children with cancer demonstrated that approximately 50% of all charges were incurred by 12.7% of patients and these patients were more likely to have AML or to have received hematopoietic stem cell transplant (HSCT) or ICU care (26). These results support the hypothesis that patients with AML are at substantial risk of treatment-related complications requiring intensive supportive care services. However, to our knowledge, there are no data on outcomes relative to need for ICU resources in pediatric patients with AML. To address this gap in outcome data, we analyzed the Pediatric Health Information Systems (PHIS) database to study a large cohort of children treated for de novo AML prior to stem cell transplant at freestanding pediatric hospitals across the United States. The primary objective of this study was to describe the prevalence of ICU care, organ failure, sepsis, and hospital mortality rates in a homogenous pediatric AML population relative to nononcology pediatric patients. Based on our institutional experience, we hypothesized that a majority of children with AML requiring ICU care would survive but that children with AML would have higher mortality in admissions requiring ICU care than children without cancer. Furthermore, we hypothesized that among those requiring ICU care, the mortality rate would be substantially higher in children with AML who had sepsis than in the corresponding group of children without cancer.

Background: CTL019 is a therapy derived from autologous T cells expressing a CD19-specific chimeric antigen receptor (CAR) that was approved by the FDA in August 2017 (tisagenlecleucel). Complete and durable remissions have been seen in the setting of pediatric and young adult patients with relapsed and refractory B cell acute lymphoblastic leukemia (ALL) (Maude NEJM 2018). Initial case reports suggested that there may be differential outcomes mediated by cytogenetic characteristics of the leukemia at CAR T cell infusion. Here, we report results from a single institution experience of 112 patients. Methods: Patients with relapsed/refractory ALL were identified as having received CTL019 either in the context of a clinical trial (NCT02906371) or commercial product (tisagenlecleucel) at Children's Hospital of Philadelphia from October 2016 to April 2019. Patients who received prior CAR T therapy were excluded. Demographic, cytogenetic, and outcome data were manually abstracted from the medical record or clinical trial datasets. High risk lesions were defined as MLL(KMT2A) rearrangements, Philadelphia-chromosome (Ph+), Ph-like, hypodiploidy, and TCF3/HLF fusion. Favorable cytogenetics were defined as the presence of hyperdiploidy or ETV6/RUNX1fusion and intermediate were defined as iAMP21, IKZF1deletion, or TCF3/PBX1. Patients were classified according to their highest risk cytogenetic characteristic and stratified by cytogenetic risk category present at CAR T cell infusion. Relapse-free survival (RFS) and overall survival (OS) was described for cohorts with more than 10 patients. Results: One hundred and twelve patients were included in the analysis, with a median age of 11 years (range 1-29) at infusion, of which 32% had had a previous allogeneic hematopoietic stem cell transplant (alloHSCT). Disease burden at the time of CTL019 infusion was heterogenous, with 61% having detectable disease in the bone marrow and 21% having more than 25% blasts by flow cytometry. Thirty-six patients (32%) had leukemias with high-risk genetic lesions at infusion, including 12 with MLL rearrangements and 18 with Ph+ or Ph-like lesions (Table 2). Thirty-one patients (28%) had hyperdiploidy or ETV6/RUNX1; 3 additional were in conjunction with high-risk cytogenetics (t(17;19) and 2 with Ph+), and 3 in the setting of intermediate-risk cytogenetics (iAmp21, TCF3/PBX1, IKZF1deletion). Figure 1 demonstrates RFS for those patients in remission at day 28 following infusion, stratified by cytogenetic risk category. Complete remission (CR) rate in the high-risk cytogenetics group was 94%. RFS at 12 months was 69% (0.50-0.82), 69% (0.40-0.86), and 67% (0.48-0.80) for non-informative, favorable, and high-risk cytogenetic groups, respectively. Figure 2 shows OS of patients infused with CTL019, again stratified by cytogenetic categories of interest, with a maximum follow-up time of 30 months. OS at 12 months was 84% (0.68-0.93) and 76% (0.56-0.88) for the non-informative and high-risk cytogenetic groups, respectively. There were no deaths in that time period for the favorable risk category. There was no statistically significant difference in RFS or OS for patients with high-risk cytogenetics. The intermediate-risk cytogenetics group (n Conclusion: Durable remissions can be achieved with CTL019 across several high-risk cytogenetic subtypes of B-ALL. Stratifying outcomes by cytogenetic risk category in this unadjusted analysis does not show a statistically significant difference in either RFS nor OS. Further investigation is needed to parse out the contribution of individual cytogenetic lesions as well as the effects of other relapse and survival risk factors at play. Figure Disclosures Rheingold: Novartis: Consultancy; Pfizer: Research Funding. Callahan:Novartis: Consultancy. Hunger:Bristol Myers Squibb: Consultancy; Amgen: Consultancy, Equity Ownership; Jazz: Honoraria; Novartis: Consultancy. Grupp:Novartis: Consultancy, Research Funding; Roche: Consultancy; GSK: Consultancy; Cure Genetics: Consultancy; Humanigen: Consultancy; CBMG: Consultancy; Novartis: Research Funding; Kite: Research Funding; Servier: Research Funding; Jazz: Other: study steering committees or scientific advisory boards; Adaptimmune: Other: study steering committees or scientific advisory boards. Maude:Kite: Consultancy; Novartis: Consultancy.

CD19-specific chimeric antigen receptor (CAR) T cell therapies have been highly effective against B cell malignancies. We previously demonstrated that differential responses to anti-CD19 CAR T cell therapy in chronic lymphocytic leukemia (CLL) are associated with early memory T cell signature in apheresed, pre-manufacturing T-cells (CAR T-cell precursors). We tested the hypothesis that the composition of CAR-T precursor cells determines clinical efficacy in adult and pediatric Acute Lymphoblastic Leukemia (ALL), Non-Hodgkin's Lymphoma (NHL), Multiple Myeloma (MM), and CLL. Apheresed T cells were engineered to express 4-1BB plus CD3-zeta-signaling CARs targeting CD19, or B cell maturation antigen (BCMA). The same 9-day manufacturing process was used for all trials. CAR T cell kinetics were monitored using a CAR gene-specific quantitative PCR assay and standard clinical response assessments were performed. Apheresed T cells from 36 CLL, 30 adult ALL, 58 pediatric ALL, 33 NHL, and 25 MM patients were immunophenotyped by flow cytometry. The CLL cohort was used to discover phenotypically distinct subpopulations associated with the two main response groups; these associations were validated in the remaining patient cohorts. Eight CD8+ T cell populations or clusters were identified using the shared-nearest-neighbor clustering method (PMID: 31178118) in the CLL cohort. T cell subsets exhibiting naive (cluster 6) or early memory (cluster 4) features were significantly enriched in responding patients, whereas an effector memory CD8 subpopulation (cluster 2) marked the non-responding patients. Mapping these clusters onto apheresed CD8+ T cells from the other four diseases showed that cluster 4 predicted response to CAR T cell therapy in NHL and myeloma but not in adult and pediatric ALL. We also examined the expression of activation-regulated molecules including HLA-DR, Ki67, and exhaustion-related molecules PD1, CTLA4, TIM3, and LAG3. A CD27+ CD8+ population expressing low level CTLA4 but none of the activation or negative regulatory molecules was significantly enriched in responding CLL patients; this cluster validated in NHL and myeloma. A similar analysis on apheresed CD4+ T cells identified an early memory population (cluster 6) enriched in CLL responders, which expresses CCR7 and CD27 but not CD45RO, CD127, CD28, or other late memory/effector molecules. However, this population did not validate in any of the other diseases. Though not statistically significant, the CD4+ clusters with the largest effect size for enrichment in responders from NHL and myeloma trials exhibited early memory T cell features and lack of HLA-DR expression, suggesting that quiescent early memory state in CD4 may also be associated with clinical responses. A separate analysis of checkpoint inhibitory receptors and activation markers in memory CD4 T cell subsets confirmed the early memory, non-activated state of this population in CLL and was validated in myeloma but none of the other diseases. In vivo activation was a shared theme in CD4+ T cells for non-responding patients as well, though these CLL-defined CD4+ apheresed T cells clusters did not significantly validate in other diseases. In summary, our data confirm and extend our predictive biomarker profile in CLL to mature B cell and plasma cell malignancies by showing that a non-cycling, non-activated early memory CD8+ T cell population in pre-manufacturing cells was validated as a biomarker in myeloma, and NHL. We also showed that responder-associated apheresed CD4+ T cells with early memory features identified in CLL after CD19 CAR T infusions are validated in myeloma after BCMA CAR T. Thus, differentiation state and in vivo activation, and potentially exhaustion, separate response groups. Our findings inform next-generation CAR T-cell manufacturing using the populations identified herein as a starting population. Disclosures Pruteanu: Novartis: Employment. Cohen:Poseida Therapeutics, Inc.: Research Funding. Garfall:Surface Oncology: Consultancy; Novartis: Research Funding; Janssen: Research Funding; Amgen: Research Funding; Tmunity: Research Funding. Milone:Novartis: Patents & Royalties: patents related to tisagenlecleucel (CTL019) and CART-BCMA; Novartis: Research Funding. Gill:Novartis: Research Funding; Tmunity: Research Funding; Carisma: Equity Ownership, Research Funding; Sensei: Consultancy; Aro: Consultancy; Fate: Consultancy. Frey:Novartis: Research Funding. Ruella:Nanostring: Consultancy, Speakers Bureau; Novartis: Patents & Royalties: CART for cancer; AbClon: Membership on an entity's Board of Directors or advisory committees. Lacey:Novartis: Patents & Royalties: Patents related to CAR T cell biomarkers; Tmunity: Research Funding; Novartis: Research Funding. Svoboda:Merck: Research Funding; BMS: Consultancy, Research Funding; Incyte: Research Funding; Pharmacyclics: Consultancy, Research Funding; Celgene: Research Funding; Kite: Consultancy; Seattle Genetics: Consultancy, Research Funding; Kyowa: Consultancy; AstraZeneca: Consultancy. Chong:Tessa: Consultancy; Novartis: Consultancy; Merck: Research Funding. Fraietta:LEK Consulting: Consultancy; Cabaletta: Research Funding; Tmunity: Research Funding. Davis:Cabaletta: Research Funding; Tmunity: Research Funding. Nasta:Rafael: Research Funding; Aileron: Research Funding; Takeda/Millennium: Research Funding; Incyte: Research Funding; Roche/Genentech: Research Funding; Merck: Consultancy; Atara: Research Funding; Debiopharm: Research Funding. Levine:CRC Oncology: Consultancy; Vycellix: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership; Novartis: Consultancy, Patents & Royalties, Research Funding; Cure Genetics: Consultancy; Avectas: Membership on an entity's Board of Directors or advisory committees; Brammer Bio: Membership on an entity's Board of Directors or advisory committees; Incysus: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy. Maude:Kite: Consultancy; Novartis: Consultancy. Schuster:Nordic Nanovector: Honoraria; Pfizer: Honoraria; AstraZeneca: Honoraria; Pharmacyclics: Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Loxo Oncology: Honoraria; Merck: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; Novartis: Honoraria, Patents & Royalties: Combination Therapies of CAR and PD-1 Inhibitors with royalties paid to Novartis, Research Funding; AbbVie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding. Stadtmauer:Celgene: Consultancy; Tmunity: Research Funding; Novartis: Consultancy, Research Funding; Takeda: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Abbvie: Research Funding. Grupp:Novartis: Consultancy, Research Funding; Roche: Consultancy; GSK: Consultancy; Cure Genetics: Consultancy; Humanigen: Consultancy; CBMG: Consultancy; Novartis: Research Funding; Kite: Research Funding; Servier: Research Funding; Jazz: Other: study steering committees or scientific advisory boards; Adaptimmune: Other: study steering committees or scientific advisory boards. Porter:Incyte: Membership on an entity's Board of Directors or advisory committees; American Board of Internal Medicine: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Glenmark Pharm: Membership on an entity's Board of Directors or advisory committees; Immunovative: Membership on an entity's Board of Directors or advisory committees; Genentech: Employment; Wiley and Sons: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. June:Novartis: Research Funding; Tmunity: Other: scientific founder, for which he has founders stock but no income, Patents & Royalties. Melenhorst:Novartis: Research Funding, Speakers Bureau; Parker Institute for Cancer Immunotherapy: Research Funding; Stand Up to Cancer: Research Funding; Incyte: Research Funding; IASO Biotherapeutics, Co: Consultancy; Simcere of America, Inc: Consultancy; Shanghai Unicar Therapy, Co: Consultancy; Colorado Clinical and Translational Sciences Institute: Membership on an entity's Board of Directors or advisory committees; Genentech: Speakers Bureau; National Institutes of Health: Research Funding.